ChIP assay was performed as we previously described15 (link). Briefly, cells were lysed by suspending in a sequence of lytic and purification buffers. DNA fragments of 300–800 bp long were obtained by treating the nuclear pellet, obtained from lytic buffers, with MNase (Cell Signaling, Beverly, MA) in digestion buffer for 6 min. DNA fragments were immunoprecipitated with specific antibodies (anti-trimethyl-Histone H3 Lys27 and rabbit IgG) at 4 °C overnight. Immunoprecipitated DNA fragments were extracted using protein-A sepharose (Sigma-Aldrich, St. Louis, MO) and purified using DNA purifying slurry (Diagenode, Denville, NJ). The amount of purified DNA was estimated by nanodrop of 200 ng purified DNA template using SYBR green chemistry as described in RT-qPCR quantification section. Promoter-specific primers used in the current study are described in Table 1 . Calculations are expressed as a percentage of the input DNA.
Dna purifying slurry
Manufactured by Diagenode
Sourced in United States
The DNA purifying slurry is a laboratory product designed for the extraction and purification of DNA samples. It is a suspension of silica-based particles that effectively bind to DNA molecules, allowing for their separation from other cellular components during the purification process.
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3 protocols using dna purifying slurry
1
ChIP Assay Protocol for Histone Modifications
2
ChIP-qPCR protocol for Histone H3K27me3
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ChIP assay was performed as described by Liu et al. [5 (link)]. Briefly, cells were lysed and treated with MNase (Cell Signaling, Beverly, MA, USA) to obtain DNA fragments of 300–800 bp that were then immunoprecipitated with target antibodies (anti-trimethyl-Histone H3 Lys27 and rabbit IgG) at 4 °C overnight. Extraction of immunoprecipitated DNA fragments using protein-A sepharose (Sigma-Aldrich) followed next prior to purification using DNA purifying slurry (Diagenode, Denville, NJ, USA). About 200 ng of purified DNA template was then amplified real-time PCR. Promoter-specific primers were used and are listed below (Table 3 ). Data was expressed as a percentage of the input DNA.
3
S. Typhimurium-induced Chromatin Immunoprecipitation
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Cells were grown in 150mm dish and infected with S. Typhimurium. Post infection, 1x10 6 cells were taken to performed ChIP according to manufacturer kit's protocol (ab500). The lysed samples were sheared using a sonicator to obtain chromatin fragments of 200-500bp with 60 amplitude 30sec on and 30sec off for 14 cycles. Sheared chromatin was then used for immuno-precipitation using ChIP grade antibodies specific for c-Fos (Invitrogen, MA5-15055), c-Jun (Invitrogen, MA5-15172), SUMOylated c-Fos, phosphorylated c-Fos (abcam. ab193365), H3 (abcam, ab1791) and IgG (abcam, ab171870) isotype acting as negative control overnight at 4°C. The immunoprecipitated DNA was then pulled using DynaBeads TM protein G (Invitrogen) through incubation at 4°C for 3 h on end-to-end rotor. The immuno-precipitated DNA and the input DNA was purified using DNA purifying slurry (Diagenode, C03080030). The purified DNA was used for performing qPCR against Ubc9, Pias1, Ccl2, Ccl19 and Ccl5 promoter. The plates were then run on qPCR machine Bio-Rad CFX 96™. The data were collected, analyzed and plotted using Graph pad prism.
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